Cryptosporidium and Giardia are important parasites due to their zoonotic potential and impact on human health, often causing waterborne outbreaks of disease. Detection of (oo)cysts in water matrices is challenging and few countries have legislated water monitoring for their presence. The aim of this study was to investigate the presence and origin of these parasites in different water sources in Northern Greece and identify interactions between biotic/abiotic factors in order to develop risk-assessment models. During a 2-year period, using a longitudinal, repeated sampling approach, 12 locations in 4 rivers, irrigation canals, and a water production company, were monitored for Cryptosporidium and Giardia, using standard methods. Furthermore, 254 faecal samples from animals were collected from 15 cattle and 12 sheep farms located near the water sampling points and screened for both parasites, in order to estimate their potential contribution to water contamination. River water samples were frequently contaminated with Cryptosporidium (47.1%) and Giardia (66.2%), with higher contamination rates during winter and spring. During a 5-month period, (oo)cysts were detected in drinking-water (<1/litre). Animals on all farms were infected by both parasites, with 16.7% of calves and 17.2% of lambs excreting Cryptosporidium oocysts and 41.3% of calves and 43.1% of lambs excreting Giardia cysts. The most prevalent species identified in both water and animal samples were C. parvum and G. duodenalis assemblage AII. The presence of G. duodenalis assemblage AII in drinking water and C. parvum IIaA15G2R1 in surface water highlights the potential risk of waterborne infection. No correlation was found between (oo)cyst counts and faecal-indicator bacteria. Machine-learning models that can predict contamination intensity with Cryptosporidium (75% accuracy) and Giardia (69% accuracy), combining biological, physicochemical and meteorological factors, were developed. Although these prediction accuracies may be insufficient for public health purposes, they could be useful for augmenting and informing risk-based sampling plans.

Biofilms containing pathogenic organisms from the water supply are a potential source of protozoan parasite outbreaks and a significant public health concern. The aim of the present study was to demonstrate the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens (WBPP) in substrate-associated biofilms (SAB) and compare it to surface water (SW) and sediments with bottom water (BW) counterparts using manual filtration and elution from low-volume samples. For scenario purposes, simulated environmental biofilm contamination was created from in-situ grown one-month-old SAB (OM-SAB) that were spiked with Cryptosporidium parvum oocysts. Samples were collected from the largest freshwater reservoirs in Luzon, Philippines and a University Lake in Thailand. A total of 69 samples (23 SAB, 23 SW, and 23 BW) were evaluated using traditional staining techniques for Cryptosporidium, and Immunofluorescence staining for the simultaneous detection of Cryptosporidium and Giardia. WBPP were found in 43% SAB, 39% SW, and 39% BW of the samples tested in the present study with SAB results reflecting SW and BW results. Further highlights were demonstrated in the potential of using low-volume samples for the detection of parasites in source water. Scanning electron microscopy of OM-SAB samples revealed a naturally-associated testate amoeba shell, while Cryptosporidium oocysts spiked samples provided a visual profile of what can be expected from naturally contaminated biofilms. This study provides the first evidence for the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens in low-volume aquatic matrices and further warrants SAB testing along with SW and BW matrices for improved water quality assessment strategies (iWQAS).

In order to establish seawater contamination by emerging protozoan parasites, we used qPCR to molecularly characterize and evaluate the parasitic burden of Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii, and Cyclospora cayetanensis in 1255 wild bivalve mollusks collected along the Tunisian coasts. T. gondii, G. duodenalis and C. cayetanensis were detected in 6.9% (99% CI=1.6–12.2%) pools of Ruditapes decussatus. None of the samples were found positive to Cryptosporidium spp.; 6.6% pools of R. decussatus were positive for T. gondii Type I, 1.6% for G. duodenalis assemblage A, and 1.6% for the association T. gondii Type I/C. cayetanensis/G. duodenalis assemblage A. R. decussatus harbored up to 77500 oocysts/sample of T. gondii, up to 395 cysts/sample of G. duodenalis, and 526 oocysts/sample of C. cayetanensis. These results provide the first evidence that the Tunisian coasts are contaminated by zoonotic protozoan parasites that can constitute a direct or indirect risk for human health.

Manila Bay is one of the major propagation sites of edible bivalves in the Philippines. Studies have shown that bivalves might be contaminated with human pathogens like the protozoan parasite Cryptosporidium, one of the major causes of gastroenteritis in the world. In this study, Cryptosporidium from four species of edible bivalves were isolated using a combination of sucrose flotation and immunomagnetic separation. Using direct fluorescent antibody test, Cryptosporidium oocysts were found in 67 out of 144 samples collected. DNA sequence analysis of the 18S rRNA gene of the isolates detected C. parvum and C. hominis (major causes of human cryptosporidiosis) and C. meleagridis (causes infection in avian species). Analysis of the 60kDa glycoprotein gene further confirmed the genotypes of the Cryptosporidium isolates. This study is the first to provide baseline information on Cryptosporidium contamination of Manila Bay where bivalves are commonly cultured.

Treatments using ozone for filter backwash water (FBW) and calcium oxide for floated residue (FR) were evaluated adopting bench-scale testing for the inactivation of Giardia spp. cysts and Cryptosporidium parvum oocysts. The protocol chosen for protozoa detection involved following the concentration step by direct centrifugation (adding ICN 7X cleaning solution at 1.0%) and purification by immunomagnetic separation (IMS). The FR treatment with calcium oxide (dosage of 23 mg CaO 100 mL⁻¹ and 3-day contact time at 25 °C) proved to be efficient, as no parasites were detected after the treatment. The reduction of calcium oxide dosage (16 mg CaO 100 mL⁻¹ and 3-day contact time at 25 °C) was insufficient to inactivate the protozoa, since potentially viable organisms were identified using propidium iodide (PI). Concerning the disinfection conditions with ozone (5-min and 10-min contact time and dosage of 10 mg O₃ L⁻¹ and 7.5 mg O₃ L⁻¹, respectively), there was complete removal of the target organisms, as no protozoa were detected after the FBW treatment. From the results obtained, the tested treatments can be considered promising alternatives for water treatment plants (WTPs). However, the costs incurred from these treatments have to be considered.

A quantitative microbial risk assessment method can be used to evaluate infections probabilities for microorganisms in a specific place. The methodology provides suitable information to generate strategies focusing on health problems. Giardia cysts (GC) and Cryptosporidium oocysts (CO) are considered emerging pathogens that can infect human and animals by ingesting contaminated food or water, where food and water are transport vehicles for these parasites. Studies for GC and CO have reported occurrences for these parasites in water up to 100%, and some of these studies documented a number of cases, about 403,000 people, infected worldwide. This review is focused on compiling the most relevant works assessing the risk for GC and CO and their presence in different water samples that are susceptible for direct and indirect human consumption. The annual risk infection probability for these parasites has been reported from different water sources, with a range between 1 × 10⁻⁶ and 1, while the world standard regulation is 1 × 10⁻⁴. The infection probability depends not only on water quality but also on water treatment implementations.

Cryptosporidium parvum oocysts may reach soil through direct deposition of human or animal fecal material, irrigation with raw wastewater or untreated effluents, and contaminated runoff. Examination of soil samples for oocyst presence is of primary importance in order to prevent secondary contamination of crops and groundwater. Several methods were proposed for oocyst recovery from soil samples; however, their efficiency was very low. In the present study, four known methods used to recover oocysts from water and fecal samples (sedimentation, sedimentation with reduced water content, sucrose floatation, water-ether separation) were compared to a method used in the past to recover bacterial spores from bottom sediments (two-phase separation). The two-phase separation technique proved to be the best method of choice resulting in a recovery average of 61.2 ± 15.6%. According to this method, the lowest and highest recoveries were 37% to 95%, respectively. Two other important outcomes were observed with the soil experimental set-up: (1) recovery efficiency is influenced by oocyst viability (high viability was directly correlated with increased recovery efficiency) and (2) high sand content of soil samples reduced oocyst recovery by its detrimental effect on oocyst viability.

Cryptosporidium and Giardia are associated with cases of water and foodborne outbreaks in the world. This study included 50 samples of surface raw water collected from two watersheds in the state of São Paulo, Brazil. The isolation of (oo)cysts was performed in accordance with the U.S. Environmental Protection Agency’s methods 1623 and genotypic characterization and quantification were carried out by Nested PCR and qPCR assays based on 18S rRNA and gdh genes, respectively. U.S. EPA 1623 method showed the presence of (oo)cysts in 40% ([Formula: see text] = 0.10 oocysts/L) and 100% ([Formula: see text] = 7.6 cysts/L) of samples from São Lourenço River, respectively, and 24% ([Formula: see text] = 0.8 oocysts/L) and 60% ([Formula: see text] = 1.64 cysts/L) of Guarapiranga Reservoir, respectively. The qPCR assay detected C. hominis/parvum in 52% (0.06 to 1.85 oocysts/L) of São Lourenço River and 64% (0.09 to 1.4 oocysts/L) of Guarapiranga Reservoir samples. Presence/absence test for Giardia intestinalis was positive in 92% of São Lourenço River and 8% of Guarapiranga Reservoir samples. The assemblage A was detected in 16% (0.58 to 2.67 cysts/L) in São Lourenço River and no positive samples were obtained for assemblage B in both water bodies. The characterization of anthroponotic species C. parvum/hominis, G. intestinalis, and assemblage A was valuable in the investigation of possible sources of contamination in the watersheds studied confirming the need of expanding environmental monitoring measures for protection of these water sources in our country.

Treatment of domestic wastewater in a pilot-scale upflow anaerobic hybrid (AH) reactor (0.9 m³) in combination with downflow hanging sponge (DHS) system (1.3 m³) was investigated. The combined system was operated at a hydraulic retention time (HRT) of 6.0 h for AH and 3.2 h for DHS system. The total process achieved a substantial reduction of CODₜₒₜₐₗ resulting in an average effluent concentration of only 39 ± 12 mg/l. Moreover, 90 ± 7 % of ammonia was eliminated in the DHS system. Nitrate and nitrite data revealed that 49 ± 3.2 % of the ammonia removal occurred through nitrification process. The removal efficiency of total coliform (TC), fecal coliform (FC), and fecal streptococci (FS) was relatively low in the AH reactor. The major portion of TC, FC, and FS was removed in the DHS system resulting to an average count of 1.7 × 10⁵ ± 1.1 × 10²/100 ml for TC, 7.1 × 10⁴ ± 1.2 × 10²/100 ml for FC, and 7.5 × 10⁴ ± 1.3 × 10²/100 ml for FS in the final effluent. Likely, the combined system was very efficient for the removal of protozoological species such as sarcodins (Entamoeba cysts), flagellates (Giardia cysts), and ciliates (Balantidium cysts). This was not the case for coccidia (Cryptosporidium oocysts), where 36.4 and 27.3 % were detected in the effluent of AH and DHS system, respectively. Only 10 % of intestinal nematode and cestode ova were recorded in the effluent of AH reactor and were completely removed in the DHS system.

With the widespread use of reclaimed water all over the world, there is a clear need to optimise its management in order to guarantee water safety. Model microorganisms (with either indicator or index function) are commonly used to assess risks related to the presence of enteric pathogens in water. Samples from five water reclamation plants located in Northeastern Spain were analysed to validate the use of three model microorganisms (Escherichia coli, somatic coliphages and spores of sulphite-reducing clostridia) as surrogates of Cryptosporidium total or infectious oocysts (TOO and IOO, respectively) in reclaimed water. Probability plots, simple and multiple linear regression and discriminant analyses were performed to assess their relationships. Results show that the detection of E. coli alone is not useful to model either the behaviour or concentrations of Cryptosporidium. However, discriminant analyses showed a high rate of correctly classified samples (91.9 %) when E. coli and somatic coliphages data were used together to predict the presence/absence of IOO. Spores of sulphite-reducing clostridia (SRC) showed parallel reduction patterns and high correlation values (r = 0.76) with reductions in TOO. Furthermore, simple regression analyses of SRC and TOO in reclaimed water showed high correlation values (r = 0.85). Therefore, at the treatment plants studied, SRC can be considered to have good indicator and index functions for TOO. From the point of view of health protection, the use of SRC together with E. coli (which is mandatory in the current Spanish regulations) would satisfy the need for improved reclaimed water management.